Microbial genes whose products act in a coordinated fashion, for example a biosynthetic pathway, are often arranged in close physical proximity to one another in the organism's genome. Such genes are said to form a gene cluster. Gene clusters are involved in the biosynthesis of complex compounds, notably the biosynthesis of microbial natural products, and in the catabolism of complex compounds (e.g. Velasco et al., J. of Bacteriology, 180(5):1063–1071; Buchan et al., Appl. and Env. Microbiol., 66(11): 4662–4672; Masai et al., J. of Bacteriology, 181(1):55–62; Ferrandez et al., J. of Biological Chemistry, 273(40), 25974–25986). Gene clusters may also provide resistance to therapeutic drugs (e.g. Schouten et al., Antimicrob Agents Chemother, 45(3):986–9). Gene clusters may constitute pathogenicity islands from various organisms (e.g. Kuroda et al., 357(9264):1225–40; Carniel E., Microbes Infect. 3(7):561–9; Nicholls et al., Mol. Microbiol 35(2):275–88).
Gene clusters are of significant interest in various fields. For example, gene clusters such as the Tn1546-like elements that are responsible for the spread of vancomycin resistance in clinical isolates of enterococci are of great interest to the medical field. The rapid identification of such clusters allows a better understanding of the spread and mechanisms of action of vancomycin resistance. Gene clusters for catabolic pathways are of interest in the field of bioremediation for the breakdown of toxic agents from contaminated environments and in the field of chemical engineering for the generation of economically valuable molecules from common, inexpensive materials. Gene clusters known as pathogenicity islands render otherwise harmless bacteria to highly pathogenic threats. For example, E.coli 0157 is a clinically important and often lethal pathogen that differs in part from the non-pathogenic E. coli K12 in that the former contains pathogenicity islands. Identification of such pathogenicity islands is of great importance to the medical field.
Natural product biosynthetic gene clusters are of significant interest in the field of combinatorial biosynthesis and metabolic engineering. Novel molecules may be made by genetic engineering of natural product biosynthetic genes. Improved methods to rapidly discover gene clusters involved in the biosynthesis of microbial natural products expands the repertoire of genes available for use in combinatorial biosynthesis and as biocatalysts and facilitates the discovery of new natural product molecules and variants of known molecules. The emergence of bacteria resistant to multiple antibiotics has led to renewed interest in isolating variants of known antibiotics and novel antibiotics, and also in identifying new genes and gene products that could serve as new targets for new or existing antibiotics.
Methods for natural product discovery have faced many challenges. Discovery efforts that focus on plant derived natural products are hampered by limited source material, typically low concentrations of active metabolite, difficulty extracting useful quantities of the natural product produced, and the fact that many secondary metabolic biosynthetic loci are expressed only under particular growth conditions (for example, pathogen infestation) that are poorly understood and may be difficult to reproduce experimentally. Discovery efforts that focus on microbial derived natural products are hampered by difficulties in cultivating the microbes; indeed most microbes have yet to be cultivated in vitro. In addition, many cultivated microorganisms are not amenable to fermentation. Furthermore many secondary metabolites are not expressed to detectable levels under in vitro conditions. Furthermore, natural products produced under in vitro conditions often vary according to the growth conditions, e.g. nutrients provided, and may not be representative of the full biosynthetic potential of the microorganism. Thus, there is a need for improved methods for discovery of gene clusters involved in the biosynthesis of natural products and for methods that do not require the cultivation, growth or fermentation of organisms.
Genome sequence of actinomycetes S. coelicolor (Bentley S. D. et al., Nature, 417, 141–147) and S. avermitilis (Omura S. et al., Proc. Natl. Acad. Sci USA 98, 12215–12220) has revealed the presence of numerous cryptic gene clusters encoding putative natural products, suggesting that well-studied strains may produce a greater number of bioactive compounds than has been detected by fermentation broth analyses. These cryptic gene clusters remain unexpressed until appropriate chemical or physical signals induce their expression. There is a need for a method of discovering gene clusters independently of expression of the genes forming the gene cluster or detection of their product.
Known methods of discovering gene clusters are often cluster-specific, and may not have broad application to smaller gene clusters or gene clusters encoding non-modular genes. In addition, many of these methods are labor-intensive, and involve sequencing significant amounts of DNA encoding genes that are not involved in the biosynthesis of the product of the target gene cluster. Because degenerate or universal probes or primers are often imperfect, natural product gene clusters may be missed. Furthermore, probes or primers may not reveal cryptic biosynthetic loci.
There is a continuing need for high throughput methods for identification of all gene clusters. There is also a need for methods for detecting natural product loci in a genome with minimal DNA sequencing, and in particular minimal sequencing of DNA encoding genes for primary metabolism. There is also a need for improved methods for detecting the biosynthetic loci for secondary metabolic pathways in an organism without having to sequence the entire genome of the organism. There is also a need for improved genomics-based methods for detecting gene clusters responsible for the biosynthesis of natural products in microbial organisms, which methods are rapid, use less reagents, are less labor-intensive, and are not dependent upon expression of the genes in the target gene clusters.